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Am J Physiol Heart Circ Physiol 282: H1709–H1716, 2002; 10.1152/ajpheart.00744.2001.

Induction of apoptosis in vascular smooth muscle cells by mechanical stretch MOHAMMAD SOTOUDEH,1,2 YI-SHUAN LI,1 NORIYUKI YAJIMA,3 CHIH-CHIEH CHANG,3 TSUI-CHUN TSOU,1 YIBIN WANG,4 SHUNICHI USAMI,1 ANTHONY RATCLIFFE,2 SHU CHIEN,1,4 AND JOHN Y.-J. SHYY3 1 Department of Bioengineering and Whitaker Institute of Biomedical Engineering, 2Advanced Tissue Sciences, La Jolla 92037; 3Division of Biomedical Sciences, University of California, Riverside 92506; and 4Department of Medicine, University of California, San Diego, La Jolla, California 92093 Received 17 August 2001; accepted in final form 10 December 2001

Sotoudeh, Mohammad, Yi-Shuan Li, Noriyuki Yajima, Chih-Chieh Chang, Tsui-Chun Tsou, Yibin Wang, Shunichi Usami, Anthony Ratcliffe, Shu Chien, and John Y.-J. Shyy. Induction of apoptosis in vascular smooth muscle cells by mechanical stretch. Am J Physiol Heart Circ Physiol 282: H1709–H1716, 2002; 10.1152/ajpheart.00744.2001.—We studied the response of porcine vascular smooth muscle cells (PVSMCs) to cyclic sinusoidal stretch at a frequency of 1 Hz. Cyclic stretch with an area change of 25% caused an increase in PVSMC apoptosis, which was accompanied by sustained activation of c-Jun NH2-terminal kinases (JNK) and the mitogen-activated protein kinase p38. Cyclic stretch with an area change of 7% had no such effect. Infection of PVSMCs with recombinant adenoviruses expressing constitutively active forms of upstream molecules that activate JNK and p38 also led to apoptosis. The simultaneous blockade of both JNK and p38 pathways with adenovirus-mediated expression of dominant-negative mutants of c-Jun and p38 caused a significant decrease (to 1/2) of the apoptosis induced by 25% cyclic stretch. The 25% stretch also caused sustained clustering of tumor necrosis factor-␣ (TNF-␣) receptor-1 and its association with TNF-␣ receptor-associated factor-2 (TRAF2). Overexpressing the wild-type TRAF-2 in PVSMCs caused an increase in apoptosis. In contrast, the expression of a dominant-negative mutant of TRAF-2 attenuated stretchinduced apoptois. These results support the hypothesis that circumferential overload under hypertensive conditions induces a clustering of death receptors that cause vascular smooth muscle cell apoptosis. c-Jun NH2-terminal kinases; p38; mechanotransduction; mechanical overload; vascular wall

A PERIODIC VARIATION in blood vessel radius in response to pulsatile pressure causes a cyclic strain on the cellular components of the vessel wall. Blood vessels respond to mechanical overload and maintain homeostasis by vascular remodeling (see Ref. 50 for review). Excessive mechanical overload can lead to the abnormal vascular changes that are seen in disease states (38, 39). Apoptosis, or programmed cell death, plays an important role in both the normal and pathological remodeling of the vessel wall (see Ref. 4 for review).

Address for reprint requests and other correspondence: J. Y.-J. Shyy, Division of Biomedical Sciences, Univ. of California, Riverside, CA 92521-0121 (E-mail: [email protected]). http://www.ajpheart.org

The application of cyclic stretch to cultured vascular smooth muscle cells (VSMCs) has been used as an in vitro experimental approach to study molecular events in response to mechanical overload. It has been shown that mechanical stretch causes the activation of multiple signaling molecules including Ca2⫹, extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinases (JNK) in the mitogen-activated protein kinase (MAPK) family (1, 15, 40); the induction of platelet-derived growth factor (PDGF), fibroblast growth factor-2, and interleukin-1 (IL-1) (5, 25, 51); the secretion of extracellular matrices such as collagen (24); and an increase in mitosis (46). A recent study by Mayer et al. (30) indicated that mechanical stretch also activated p38, another MAPK member. Overexpression of a dominant-negative mutant of small GTPase Rac or MAPK phosphatase-1 in VSMCs completely eliminated mechanical stress-activated p38 and abolished mechanical stress-induced apoptosis (30). However, the upstream mechanotransduction events leading to the p38 activation are still unclear. Signaling cascades mediated by MAPKs play important roles in regulating cellular functions including apoptosis. JNK and p38, which have been suggested to be involved in environmental stress-induced apoptosis (11, 23, 45), can be activated by various apoptotic stimuli such as IL-1, tumor necrosis factor (TNF)-␣, ultraviolet (UV) irradiation, and ␥-irradiation (7, 13, 20, 22). Overexpression of MAPK kinase (MKK)-7 and MKK-3, the respective upstream kinases that activate JNK and p38, leads to sustained activation of JNK and p38 as well as cell apoptosis (22, 37, 47–49, 52). TNF-␣ receptor-1 (TNFR-1) is one of the death receptors responsible for apoptotic signaling (see Ref. 34 for review). UV irradiation and osmotic pressure can cause TNFR-1 clustering and JNK activation independent of the ligand TNF-␣ (41). TNF-␣ receptor-associated factor-2 (TRAF-2) has been identified as an important docking protein that links TNFR-1 to its downstream signaling molecules such as JNK and p38 (36). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society

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We hypothesize that death receptors such as TNFR-1 are a critical factor in mechanical stretchinduced apoptosis. In this report, we studied the effects of cyclic stretches on cultured porcine VSMCs (PVSMCs) with area changes of 7 and 25% with the aim of simulating the difference in wall stresses experienced by the cells under normal and elevated pressure levels, respectively. The results show that an area change of 25% but not 7% causes the clustering of TNFR-1, sustained activations of JNK and p38, and the ensuing VSMC apoptosis. METHODS

Cell culture and cyclic stretch experiments. A stretch apparatus (44) was used to apply uniform, sinusoidal, cyclic stretch to PVSMCs that had been cultured on silicon elastic membranes (Specialty Manufacturing; Saginaw, MI) coated with fibronectin (2.5 ␮g/cm2; Sigma, St. Louis, MO). The amplitude of the cyclic stretch was 7 or 25%, and the frequency was 1 Hz. The cells were maintained in Dulbecco’s modified Eagle’s media (DMEM) containing 10% fetal bovine serum (FBS). Adenoviral infection of PVSMCs. Recombinant adenoviruses expressing the constitutively active forms of MKK-3 and MKK-7 and the dominant-negative mutants of c-Jun (DN-c-Jun) and p38 (DN-p38) were described previously (21, 48, 49). In brief, the various cDNAs were cloned into the appropriate sites of the pAdv/Rous sarcoma virus shuttle vector. The recombinant adenoviruses were then generated by homologous recombination between the plasmid pJM17 and shuttle plasmids in 293 human embryo kidney cells. Confluent PVSMCs were infected with the recombinant adenoviruses at a concentration of 40 plaque-forming units (pfu) per cell for 12 h. The infected cells were allowed to recover for an additional 72 h before being subjected to various experiments. Kinase activity assays. JNK activity assays were performed according to the procedures previously described (7). Static or stretched PVSMCs were lysed and immunoprecipitated with an anti-JNK (Santa Cruz Biotechnology; Santa Cruz, CA). The kinase reaction was initiated by adding [␥-32P]ATP (ICN; Irvine, CA) and a glutathione S-transferase (GST)-c-Jun-(1–79) fusion protein. The phosphoproteins were separated via SDS-PAGE and detected by autoradiography. The kinase activity of p38 was assessed by using the same procedures as those used for JNK except that an antip38 (Santa Cruz Biotechnology) was used for immunoprecipitation and myelin basic protein (MBP) was used as the substrate in the kinase reaction. Ligation-mediated polymerase chain reaction. The genomic DNA was isolated using the Puregene kit (Gentera Systems; Minneapolis, MN). DNA fragmentation analysis was performed using a ligation-mediated polymerase chain reaction (LM-PCR) ladder-assay kit (Clontech; Palo Alto, CA). Briefly, 1 ␮g of genomic DNA from each experiment was used for adapter ligation. For each LM-PCR reaction, 150 ng of adapterligated DNA was used. The PCR products were separated on a 1.2% agarose gel. A set of the En-2 primers was used as the internal control. Flow cytometry analysis. PVSMCs were trypsinized, washed with cold PBS, resuspended in 1⫻ binding buffer (105 cells/100 ␮l; R&D Systems; Minneapolis, MN), and transferred to a glass tube. To each tube, 10 ␮l each of annexin V and propidium iodide were added for 15 min of incubation. AJP-Heart Circ Physiol • VOL

The samples were then subjected to flow cytometry analyses using a FACScan (Becton Dickinson; San Jose, CA). Immunostaining. For immunostaining of TNFR-1, PVSMCs were fixed with 3% formaldehyde, incubated with an antiTNFR-1 (R&D Systems), and subsequently incubated with a FITC-conjugated anti-goat IgG (Santa Cruz Biotechnology). The specimens were observed and photographed with a Nikon Diaphot 300 inverted microscope. For studying the role of TRAF-2 in apoptosis, PVSMCs were transfected with Myc-TRAF-2 or Myc-TRAF-2(⫺) plasmid using the GenePORTER transfection reagent (Gene Therapy Systems; San Diego, CA). After transfection for 48 h, the specimens were subjected to immunostaining first with an anti-c-Myc (Santa Cruz Biotechnology) and then by the tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody to identify the transfected cells. The nuclear chromatin of all cells was detected by fluorochrome bisbenzimide trihydrochloride (Hoechst 33258) staining (Boehringer Mannheim; Indianapolis, IN). ELISA assay. Conditioned media collected from stretch experiments or static controls were centrifuged to remove cell debris, and the volumes of the various samples were calibrated. The TNF-␣ concentration in the media was measured with the use of the ChemiKine TNF-␣ ELISA kit (Chemicon; Temecula, CA). Briefly, 100 ␮l of the media were applied to immunoplate precoated with anti-human TNF-␣ monoclonal antibody. Polyclonal anti-TNF-␣ was then added to detect the immobilized TNF-␣ in the sample. The conjugation of antiTNF-␣ with its antigen was visualized using goat anti-rabbit IgG conjugated with alkine phosphatase, and optical density was then measured. Statistics. All experiments were performed independently at least three times. Student’s t-test and ANOVA were used to determine statistical significance. RESULTS

Cyclic stretch of 25% induces PVSMC apoptosis and JNK and p38 activation. PVSMCs cultured on fibronectin-coated membranes were kept as static controls or subjected to 7 or 25% cyclic stretch up to 72 h before we performed detection of DNA fragmentation. Cyclic stretch with 25% area change exerted an apoptotic effect on PVSMCs. The cells subjected to 25% cyclic stretch for 24, 48, and 72 h showed increasing degrees of DNA fragmentation over time, which indicates the progression of apoptosis (Fig. 1A). In contrast, apoptosis did not increase significantly in either static controls or cells subjected to 7% cyclic stretch (data not shown). DNA fragmentation was seen in PVSMCs treated with sodium nitroprusside (SNP), which is known to induce apoptosis in VSMCs (12) and thus served as a positive control. Our findings that mechanical stretch induces apoptosis of PVSMCs are similar to those reported for mouse, rat, and human VSMCs (30). Because 25% but not 7% cyclic stretch caused PVSMC apoptosis, we examined the effects of these two magnitudes of stretch on the activities of JNK and p38. PVSMCs were kept as static controls (C) or subjected to 7 or 25% cyclic stretch (S) for 4 h before receiving JNK and p38 kinase activity assays. As shown in Fig. 1, B and C, the kinase activities of JNK and p38 in cells subjected to 7% cyclic stretch for 4 h were comparable to those in the respective static con-

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trols. In contrast, 25% cyclic stretch for 4 h significantly increased the activity of JNK (S/C ratio ⫽ 2.1 ⫾ 0.4) and p38 (S/C ratio ⫽ 1.7 ⫾ 0.1). We further studied the temporal responses of JNK and p38 to 25% cyclic stretch (Fig. 2). The JNK and p38 activities increased significantly at 1 h, and the degree of induction was sustained at essentially the same levels for at least 48 h. PVSMC apoptosis by cyclic stretch is mediated through JNK and p38. To determine the effect of the sustained activation of JNK and p38 on the PVSMC apoptosis induced by cyclic stretch, we infected the cells with recombinant adenoviruses that express ac-

Fig. 1. Cyclic stretch with an area change of 25% but not 7% induces apoptosis of porcine vascular smooth muscle cells (PVSMCs) and activation of c-Jun NH2-terminal kinases (JNK) and mitogen-activated protein kinase (MAPK) p38. A: DNA fragmentation assay showing that 25% cyclic stretch induces PVSMC apoptosis. Genomic DNAs isolated from static, stretched, or sodium nitroprusside (SNP)treated PVSMCs were subjected to ligation-mediated polymerase chain reaction (LM-PCR) analysis and subsequent agarose gel electrophoresis. PVSMCs were subjected to 7 or 25% cyclic stretch or kept as static controls for 4 h. Kinase activities of JNK (B) and p38 (C) were assayed using glutathione S-transferase (GST)-c-Jun and myelin basic protein (MBP) as respective substrates (arrows). Relative (stretched-to-static ratio) kinase activities (means ⫾ SD) are plotted. * P ⬍ 0.05, significant difference between stretched samples and static controls. AJP-Heart Circ Physiol • VOL

Fig. 2. Cyclic stretch of 25% causes sustained activation of JNK and p38 in PVSMCs. PVSMCs were subjected to 25% area stretch for the indicated time points or kept as static controls. Kinase assays for JNK and p38 were performed using the same procedures described in Fig. 1. Phosphorylated GST-c-Jun and MBP are indicated by arrows. Relative (stretched-to-static ratio) kinase activities (means ⫾ SD) are plotted for JNK (A) and p38 (B). * P ⬍ 0.05, significant difference between stretched samples and static controls.

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tive forms of MKK-3 (AdMKK3) or MKK-7 (AdMKK7), which are the respective upstream kinases that activate p38 and JNK (49, 52). DNA fragmentation assays showed that PVSMC apoptosis was induced by infection with AdMKK3 or AdMKK7, but not in cells infected with AdLacZ encoding ␤-galactosidase (Fig. 3A). These results demonstrate that sustained activation of JNK or p38 in PVSMC is sufficient for the induction of apoptosis. We next examined whether JNK and/or p38 pathways were critical for the cyclic stretch-induced cell death. Because of the inefficient expression of the JNK dominant-negative mutant JNK(K-R) in our system, we used DN-c-Jun to block the JNK pathway. PVSMCs were infected with recombinant adenoviruses express-

Fig. 3. JNK and p38 mediate the stretch-induced PVSMC apoptosis. PVSMCs were infected with the replication-deficient adenoviruses AdLacZ, AdMKK3, or AdMKK7 [40 plaque-forming units (pfu)/cell] for 12 h (A). AdMKK3 and AdMKK7 are the recombinant adenoviruses expressing MAPK kinase (MKK)-3 and MKK-7, the active forms of the molecules directly upstream to p38 and JNK, respectively. Infected cells were cultured in DMEM containing 10% fetal bovine serum (FBS) for 72 h before being subjected to DNA fragmentation analysis. Apoptosis is indicated by DNA fragmentation. Dominant-negative mutants of c-Jun (DN-c-Jun) and p38 (DN-p38) attenuate stretch-induced apoptosis (B). PVSMCs were infected with AdLacZ, DN-c-Jun, DN-p38, or a combination of DN-c-Jun and DN-p38 (40 pfu/cell) for 12 h. After 25% stretch for 48 h, the cells were stained with propidium iodide, fixed, and subjected to flowcytometry analysis. Ratios of cell death are plotted (means ⫾ SD). * P ⬍ 0.05, significant differences between DN-c-Jun ⫹ DN-p38 group and AdLacZ control. AJP-Heart Circ Physiol • VOL

ing AdLacZ, DN-c-Jun, DN-p38, or a combination of DN-c-Jun and DN-p38. The cells were kept as static controls or subjected to 25% stretch for 48 h and subsequent flow cytometry analysis. As shown in Fig. 3B, the application of 25% cyclic stretch for 48 h to AdLacZinfected PVSMCs increased cell death to result in an S/C ratio of 3.0 ⫾ 0.4 (i.e., an increase of 200% above the static controls). Infection of PVSMCs with either DN-p38 or DN-c-Jun alone did not significantly attenuate the level of cell death. However, coinfection of both DN-c-Jun and DN-p38 significantly reduced cell death to an S/C of 1.5 ⫾ 0.3 (i.e., only 50% greater than that in static controls). Hence, the blockade of both JNK and p38 pathways reduced the stretch-induced cell death from 200 to 90%. TNFR-1 clustering is induced by 25% but not 7% cyclic stretch. We hypothesized that TNFR-1 clustering in the cell membrane is involved in the mechanotransduction mechanism, by which 25% cyclic stretch induces PVSMC apoptosis. This is based on previous reports (36, 41) that show that TNFR-1 is a cell surface death receptor and that its clustering, even in the absence of its ligand, can modulate the JNK-regulated apoptosis. PVSMCs cultured on fibronectin-coated membranes were kept as static controls or subjected to 7 or 25% cyclic stretch. In parallel experiments, static cells were treated with TNF-␣ as a positive control. Immunostaining with anti-TNFR-1 showed that the application of 25% cyclic stretch caused clustering of TNFR-1 in PVSMCs (Fig. 4A). This stretch-induced TNFR-1 clustering was similar to that seen in cells treated with TNF-␣, a condition that is known to cause apoptosis (22). In contrast, neither static controls nor cells subjected to 7% stretch showed TNFR-1 clustering. ELISA assay showed no increased concentration of TNF-␣ in the media collected from 25% stretch experiments (Fig. 4B), thus the stretch-induced clustering was not due to a TNF-␣ autocrine stimulation. TRAF-2 mediates the stretch-induced PVSMC apoptosis. To investigate further the signaling events downstream of the stretch-induced TNFR-1 clustering, we studied whether 25% cyclic stretch caused the TNFR1/TRAF-2 association in PVSMCs that was previously found in PC60 cells stimulated by TNF-␣ (6). Immunoprecipitation of cell lysates from static or stretched PVSMCs with polyclonal anti-TNFR-1 that received subsequent immunoblotting with anti-TRAF-2 revealed that 25% cyclic stretch increased the association of TNFR-1 with TRAF-2 (Fig. 5) compared with the static controls. The stretch-induced association was found at 1 h and was sustained for at least 48 h. These results suggest that the TNFR-1/TRAF-2 association is an upstream signaling event in response to 25% cyclic stretch that leads to PVSMC apoptosis. To investigate the role of the TNFR-1/TRAF-2 association in PVSMC apoptosis, we transfected PVSMCs with Myc-TRAF-2 encoding the wild-type TRAF-2. The transfected cells were identified by immunostaining with an anti-Myc antibody, and apoptosis was detected by Hoechst staining to reveal the nuclear condensation. The cells that overexpressed Myc-TRAF-2 (indi-

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TRAF-2 attenuated the stretch-induced apoptosis. Collectively, the results presented in Figs. 5 and 6 suggest that TNFR-1 clustering and the ensuing association of TRAF-2 lead to PVSMC apoptosis induced by the 25% cyclic stretch. DISCUSSION

We studied the effects of equibiaxial cyclic stretch on PVSMCs. The mechanical overload due to 25% area change caused the clustering of TNFR-1 and recruited the adapter TRAF-2. Our data indicate further that the TNFR-1/TRAF-2 association can lead to the sustained activation of JNK and p38 and stretch-induced VSMC apoptosis. In contrast, 7% stretch had little effect on TNFR-1 clustering nor could it induce apoptosis. The cyclic stretch of arterial vessels due to the normal pulsatile pressure is between 2 and 18% (3, 8). It has been reported that brachial artery diameter was significantly increased in essential hypertensive patients compared with normal subjects (42). Similar positive correlation between the mean pressure of the retinal artery and its diameter was observed in hypertensive patients (17). In these reports, it was shown that hypertension can cause an increase in major artery and retinal artery diameters by 15 and 35%, respectively (17, 42). Thus the effects of 25% stretch may be relevant to the pathophysiology of hypertension, whereas

Fig. 4. Cyclic stretch of 25% but not 7% causes the clustering of tumor necrosis factor (TNF)-␣ receptor-1 (TNFR-1) (A). PVSMCs were subjected to cyclic stretches or kept as static controls for 48 h. In the TNF-␣-positive control group, the cells were treated with TNF-␣ (400 ng/ml). After stretching or TNF-␣ treatment, the specimens were fixed and stained using polyclonal anti-TNFR-1 and then FITC-conjugated anti-goat IgG. Fluorescence microscopy revealed the clustering of TNFR-1 in cells subjected to 25% stretch and those treated with TNF-␣ but not in cells subjected to 7% stretch or in static control cells. B: ELISA assay for TNF-␣ in the media collected from static or cyclically stretched cells. The optical density (OD) measurements from the ELISA reader are plotted (means ⫾ SD; values are averaged from 4 independent experiments). * P ⫽ no statistical difference among any of the stretched samples and static controls.

cated by red fluorescence of rhodamine staining in Fig. 6A) underwent apoptosis under static conditions. In contrast, the nontransfected cells showed no sign of apoptosis. TRAF-2(⫺) is a dominant-negative mutant of TRAF-2 in which the NH2 terminus has been truncated, and it has been shown to inhibit the CD27induced JNK (14) and latent membrane protein 1-induced p38 (9). We transfected PVSMCs with TRAF2(⫺) or LacZ in the control groups and subjected the transfected cells to 25% cyclic stretch and subsequent immunostaining to detect PVSMC apoptosis. As shown in Fig. 6B, 36 ⫾ 6% of the LacZ-transfected cells underwent apoptosis after mechanical stretch for 24 h, but only 22 ⫾ 4% of the TRAF-2(⫺)-transfected cells were apoptotic, which indicates that the blockade of AJP-Heart Circ Physiol • VOL

Fig. 5. Cyclic stretch of 25% increases the association of TNFR-1 with TNF-␣ receptor-associated factor-2 (TRAF-2) in PVSMCs. PVSMCs were subjected to cyclic stretch for the indicated time points or kept as static controls. After stretching, the cells were lysed and subjected to immunoprecipitation (IP) with a polyclonal antiTNFR-1 and subsequent immunoblotting (IB) with a polyclonal antiTRAF-2. An increase of the coimmunoprecipitated TRAF-2 in the stretched samples indicates an increase of its association with TNFR-1 (top). Relative association levels are compared to static controls for various time points (middle). Values are means ⫾ SD. * P ⬍ 0.05, significant difference between stretched samples and static controls. Equal loading of the samples is indicated by the ␣-tubulin protein expression detected by immunoblotting (bottom).

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Fig. 6. TRAF-2 mediates the stretch-induced PVSMC apoptosis. PVSMCs were transiently transfected with plasmid expressing the wild-type Myc-TRAF-2 and subjected to double staining (A). Transfected cells were identified by immunostaining with anti-Myc monoclonal antibody (MAb) and subsequent anti-mouse tetramethylrhodamine isothiocyanate-conjugated secondary antibody as shown by the red fluorescence of rhodamine. Nuclear chromatin of all cells was detected by Hoechst staining, which indicates that only these transfected cells underwent apoptosis. PVSMCs were transiently transfected with plasmid-expressing LacZ or AdTRAF-2(⫺), and the transfected cells were subjected to 25% cyclic stretch for 24 h (B). Double immunostaining procedure was the same as in A except that the LacZ-positive cells were identified by anti-LacZ MAb. Percentages of transfected cells that were apoptotic after mechanical stretch are shown. Values are means ⫾ SD of 3 experiments. * P ⬍ 0.05, significant difference between LacZ-transfected controls and TRAF2(⫺)-transfected cells.

the 7% stretch can be considered to be within the physiological range. Under basal culture conditions and after the addition of transforming growth factor-␤1 or pentoxifylline, VSMCs cultured from spontaneously hypertensive rats have a higher level of apoptosis than those from normotensive controls (16). It should be noted that arterial compliance is reduced after vascular remodeling under hypertensive conditions (31) and that reduced compliance would lead to a greater wall stress with less lumen distension in response to pressure elevation. In our study, the PVSMCs subjected to cyclic stretch also experienced a concurrent cyclic stress, and the stress-strain relation is not the same as that in the remodeled vessel. In this context, our results are more relevant to the effects of an increase in blood pressure on unremodeled normal vessels, e.g., that resulting from neurohumoral stimulation. Another example of clinical relevance of the 25% stretch is the expansion of a venous graft after its introduction into an artery. In a recent study by Moore et al. (33) using an end-to-end anastomosed rat vein graft model, it was reported that the tensile strain changes were AJP-Heart Circ Physiol • VOL

sustained for up to 30 days, and that significant cell death could be observed for up to 10 days. Mechanical stretch with magnitudes of ⱕ15% has been shown to cause a transient activation of JNK or ERK in several cell types including rat aortic smooth muscle cells (15, 28, 29, 53). The transient nature of JNK and p38 activation after 7% stretch supports these studies. Our new finding is that 25% stretch caused a sustained activation of JNK and p38 and the occurrence of apoptosis. Thus this study demonstrated that the temporal responses of MAPKs in cardiovascular cells elicited by different magnitudes of stretch play an important role in determining cell fate. This notion is supported by the finding that sustained activation of JNK by the disruption of microtubules causes apoptosis, whereas laminar flow induces transient activation of JNK without noticeable apoptosis (19). The different temporal responses of MAPKs in cardiovascular cells elicited by mechanical stretch versus laminar shear may result from different upstream signal transduction pathways. In analyzing the roles of JNK and p38 in mediating the VSMC apoptosis induced by 25% stretching, we found that apoptosis was not significantly attenuated individually by the dominant mutants DN-p38 or DNc-Jun, but was significantly reduced by the coinfection of both. These results suggest that multiple signaling pathways are involved in stretch-induced cell death, and that JNK and p38 provide parallel and converging pathways that mediate stretch-induced apoptosis. It has been suggested that the transient JNK activation in rat aortic smooth muscle cells is mediated through an autocrine stimulation of purinoceptors by ATP and adenosine (15) or by Ras and Rac GTPases (28). Stretch activation of ERK in cardiac myocytes is regulated by Rho GTPase (1). In addition, Rac has also been shown to be involved in stretch-induced VSMC apoptosis, because the negative mutant of Rac blocked the induced apoptosis (30). The Rac-induced apoptosis has been suggested to be related to an increase in the synthesis of the death receptor FasL (10). Taken together with our finding that TNFR-1 augments its association with TRAF-2 in response to mechanical overstretch, it is likely that both death-receptor-engaged pathways are involved in the stretch-induced VSMC apoptosis. An alternative possibility is that Rac may also act downstream of TRAF-2 (32) to mediate the stretch-induced MAPK activities. The mechanism by which the sustained activation of JNK and p38 leads to apoptosis in response to 25% cyclic stretch remains unclear. There is evidence that JNK and p38 can mediate apoptosis through the regulation of MAPK p53 (11, 20). In cardiac myocytes, p53 transcriptional activity can be increased by stretching (26), and a negative mutant of p53 has been shown to inhibit stretch-induced apoptosis (27). Further studies are needed to identify the role of p53 in 25% cyclic stretch-induced MAPK activation and VSMC apoptosis. The clustering of TNFR-1 induced by 25% cyclic stretch is likely to be mediated through a perturbation of the cell membrane, which was previously suggested

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to be the cause of stretch activation of PDGF receptors (18). The TNF-␣-induced apoptosis is mediated through the oligomerization of TNFR-1, i.e., its cognate receptor (2), and the association of TNFR-1 with its adapter protein, TRAF-2 (37). The recruited TRAF-2 can interact with apoptosis signal-regulating kinase-1 (ASK-1) to activate JNK and p38 (37, 43). The stretchinduced association of TNFR-1 with TRAF-2 indicates that mechanical stimuli (e.g., stretch) and chemical stimuli (e.g., TNF-␣) share similar signaling pathways to induce cell apoptosis. Our finding that overexpression of TRAF-2 was sufficient for PVSMC apoptosis (Fig. 6A) further indicates that TNFR-1/TRAF-2 association can activate the signaling pathways leading to apoptosis. UV and osmotic stress also induce the clustering of TNFR and the subsequent activation of JNK, which are independent of TNF-␣. Thus the clustering of death receptors such as TNFR-1 can be a common theme of cellular responses to environmental stresses. In summary, our results suggest the following sequence of events for the apoptosis induced by 25% cyclic stretch: excessive stretching causes a clustering of TNFR-1 and its association with TRAF-2, which leads to a sustained activation of JNK and p38 to induce the death of VSMCs. The authors thank Dr. Gang Jin and Stephen Hawley for excellent material and technical support. This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-56707, HL-60789 (to J. Y.-J. Shyy), HL19454, HL-43026, and HL-64382 (to S. Chien), by US Government support under Cooperative Agreement 70NANB7H3060 awarded from the National Institute for Standard Technology, and by a gift from Dr. Shi H. Huang of the Chifon Group. J. Y.-J. Shyy is an Established Investigator of the American Heart Association. REFERENCES 1. Aikawa R, Komuro I, Yamazaki T, Zou Y, Kudoh S, Zhu W, Kadowaki T, and Yazaki Y. Rho family small G proteins play critical roles in mechanical stress-induced hypertrophic responses in cardiac myocytes. Circ Res 84: 458–466, 1999. 2. Ashkenazi A and Dixit VM. Death receptors: signaling and modulation. Science 281: 1305–1308, 1998. 3. Bergel DH. The static elastic properties of the arterial wall. J Physiol 156: 445–447, 1961. 4. Best PJ, Hasdai D, Sangiorgi G, Schwartz RS, Holmes DR Jr, Simari RD, and Lerman A. Apoptosis: basic concepts and implications in coronary artery disease. Arterioscler Throm Vasc Biol 19: 14–22, 1999. 5. Cheng GC, Briggs WH, Gerson DS, Libby P, Grodzinsky AJ, Gray ML, and Lee RT. Mechanical strain tightly controls fibroblast growth factor-2 release from cultured human vascular smooth muscle cells. Circ Res 80: 28–36, 1997. 6. Declercq W, Denecker G, Fiers W, and Vandenabeele P. Cooperation of both TNF receptors in inducing apoptosis: involvement of the TNF receptor-associated factor binding domain of the TNF receptor 75. J Immunol 161: 390–399, 1998. 7. Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin M, and Davis RJ. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76: 1025–1037, 1994. 8. Dobrin PB. Mechanical properties of the arterial wall. Physiol Rev 58: 397–460, 1978. 9. Eliopoulos AG, Gallagher NJ, Blake SM, Dawson CW, and Young LS. Activation of the p38 mitogen-activated protein kinase pathway by Epstein-Barr virus-encoded latent membrane protein 1 coregulates interleukin-6 and interleukin-8 production. J Biol Chem 274: 16085–16096, 1999. AJP-Heart Circ Physiol • VOL

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